Molecular marker related to low oxygen stress resistance of litopenaeus vannamei and application of molecular marker

Abstract

According to the molecular marker related to the low-oxygen stress resistance of the litopenaeus vannamei, an SNP molecular marker is cloned from a PGM2 gene, and the SNP marker is a 357th basic group T or C from the 5'end of a first nucleotide sequence shown in a sequence table. According to statistics, the low dissolved oxygen tolerance of the litopenaeus vannamei with the genotype of TT at the site of the SNP marker is obviously higher than that of the litopenaeus vannamei with the genotype of TC, so that the low dissolved oxygen tolerance of the litopenaeus vannamei can be effectively determined by detecting the SNP site of the litopenaeus vannamei; the SNP marker is closely related to the low dissolved oxygen resistance character of the litopenaeus vannamei, and can be effectively used for molecular marker-assisted breeding of the litopenaeus vannamei.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic animal genetics and molecular marker-assisted selection breeding, and in particular to a SNP marker of a hypoxia stress-related gene of white shrimp Penaeus vannamei, a detection primer and an application thereof. Background Art

[0002] Litopenaeus vannamei, commonly known as the whiteleg shrimp, is an important commercial aquaculture species, widely distributed in tropical and subtropical regions. Its rapid growth, high-quality meat, and strong resistance to stress have made it a popular aquaculture species among farmers and consumers. According to the 2023 China Fisheries Statistical Yearbook, my country's total aquaculture production of Litopenaeus vannamei reached 1.34 million tons in 2022, accounting for 68% of total crustacean aquaculture production, making it a key economic aquaculture target in my country. During the cultivation of Litopenaeus vannamei, dissolved oxygen concentrations must be maintained above 4 mg / L to ensure that their metabolic needs are fully met and promote healthy growth. Currently, Litopenaeus vannamei is primarily farmed using semi-intensive and intensive methods. However, in practice, insufficient dissolved oxygen levels frequently occur due to factors such as the decomposition of leftover bait, plankton respiration, and high-density farming. This not only impacts the normal growth of the shrimp but also poses potential economic risks to the aquaculture industry. Effectively improving the germplasm resources of Penaeus vannamei and cultivating new varieties of Penaeus vannamei with strong tolerance to low dissolved oxygen are particularly important for the sustainable development of the shrimp farming industry.

[0003] Single nucleotide polymorphisms (SNPs) refer to variations in a single nucleotide in the genome. This type of genetic variation is extremely common in the human genome, with an average of one SNP occurring every 500 to 1000 base pairs. SNPs are characterized by high genetic stability, abundant loci, and a wide distribution. They can occur in both coding and non-coding regions of genes and may affect gene function and regulation. SNPs located in gene coding regions may lead to changes in protein structure and function, becoming a potential direct cause of mutation or disease in organisms. Therefore, as a common form of genetic variation, SNPs can effectively reflect genetic differences between individuals and provide important molecular markers for the study of genes related to hypoxia stress.

[0004] The present invention aims to provide a SNP marker associated with low dissolved oxygen stress resistance. The SNP marker is identified through candidate SNP association analysis based on the results of a previous genome-wide association analysis using genome resequencing data. A primer pair for detecting the SNP marker of the present invention is also provided, as well as a method for marker-assisted genetic breeding of low dissolved oxygen stress resistance in Litopenaeus vannamei. Summary of the Invention

[0005] The present invention aims to provide a molecular marker associated with the hypoxia tolerance trait of Penaeus vannamei. This molecular marker can be applied to molecular marker-assisted selection breeding for the hypoxia tolerance trait of Penaeus vannamei, providing useful information for association studies of the hypoxia tolerance trait and accelerating the breeding process of superior stress-resistant varieties of Penaeus vannamei.

[0006] The present invention is achieved through the following technical solution: a SNP marker related to low dissolved oxygen tolerance of white shrimp Litopenaeus vannamei is cloned from the PGM2 gene to obtain a SNP molecular marker, the SNP marker is the 357th base T or C from the 5' end of the first nucleotide sequence shown in the sequence table.

[0007] Another object of the present invention is to provide a primer pair for detecting the SNP marker described in the present invention, wherein the primer pair is specifically primers PGM2-F and PGM2-R, which are designed using the mRNA sequence of the PGM2 gene of white shrimp (Penaeus vannamei) (Gen Bank Accession: NO. XM_070132845) as a template using Primer 5.0 software.

[0008] The nucleotide sequences of the primer pairs are shown in SEQ ID NO.1 and SEQ ID NO.2:

[0009] (1) SEQ ID NO.1: GCCACAGGTTTGCCAGAT;

[0010] (2)SEQ ID NO.2: CCAGAGGGTCAGTAAGTCG

[0011] The primer pair is used to perform PCR amplification on the fragment where the SNP marker is located, and then detect the SNP marker by sequencing to determine the genotype of the SNP marker site of the tested shrimp. The primer pair can also be used as one of the components of a kit for detecting the SNP marker.

[0012] The third object of the present invention is to provide a method for screening SNP markers related to the low dissolved oxygen tolerance trait of Litopenaeus vannamei, the specific steps of which are as follows:

[0013] (1) Using the genomic DNA of the shrimp Litopenaeus vannamei after the low dissolved oxygen stress experiment as a template, PCR amplification was performed using the above-mentioned primer pair, namely primer PGM2-F and primer PGM2-R, to obtain an amplified fragment with a length of 300 bp;

[0014] (2) sequencing the amplified fragment obtained in step (1) to obtain a partial sequence of the PGM2 gene, performing BLAST comparison on the sequence results to screen out base mutation sites, i.e., SNP sites;

[0015] (3) The chi-square test of SPSS24.0 software was used to conduct a correlation analysis between the tolerance to low dissolved oxygen and the genotype of Penaeus vannamei.

[0016] The method for extracting the genomic DNA in step (1) is not particularly limited and can be performed using any known genomic DNA extraction method or kit. The conditions for PCR amplification of the genomic DNA are also not particularly limited and can be optimized and selected by those skilled in the art.

[0017] Direct sequencing is a detection technology with the highest accuracy, strong flexibility, high throughput, and short detection cycle. This method only requires designing a pair of primers on either side of the SNP site, amplifying the resulting product, and then directly detecting the SNP site through sequencing. Therefore, the present invention preferably uses direct sequencing methods for SNP marker detection.

[0018] The SNP sites obtained through the steps described in the above technical solution can be applied to the breeding process of low dissolved oxygen tolerance traits of Penaeus vannamei. The specific method is as follows: (1) Sampling the candidate population of Penaeus vannamei breeding, and the sampling sites are selected from the tentacles or feet, which are less affected and can recover quickly. The sampled shrimp are individually marked using the eyestalk ring or fluorescent labeling method. (2) The PCR products of the samples are directly sequenced using the above primers to obtain SNP site typing information. (3) Combined with the typing information of other sites related to stress resistance traits, individuals with the TT genotype at the 357th SNP site in the PGM2 gene are preferentially selected as breeding parents for low oxygen tolerance of Penaeus vannamei for large-scale breeding, and individuals with the TC genotype at the SNP site are avoided as breeding parents for large-scale breeding.

[0019] The beneficial effects of the present invention are:

[0020] (1) Statistics show that the tolerance of Penaeus vannamei with the genotype TT at the site of the SNP marker of the present invention to low dissolved oxygen is significantly higher than that of Penaeus vannamei with the genotype TC. Therefore, by detecting the above SNP site of Penaeus vannamei, its low dissolved oxygen tolerance can be effectively determined; the SNP marker of the present invention is closely related to the low dissolved oxygen tolerance trait of Penaeus vannamei and can be effectively used in molecular marker-assisted breeding of Penaeus vannamei.

[0021] (2) The technical solution of the present invention can be used to select early-stage Penaeus vannamei breeding materials according to actual breeding needs, effectively improving breeding efficiency and accuracy, and improving the performance of Penaeus vannamei breeding populations, thereby accurately and efficiently breeding Penaeus vannamei varieties with strong stress resistance;

[0022] (3) The method is highly practical, has no specific requirements for genomic DNA extraction, sequencing methods, and individual markers, and has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the solutions in this application, a brief introduction will be given below to the drawings required for use in the description of the embodiments of this application. Obviously, the drawings described below are some embodiments of this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] Figure 1 In the embodiment, the haplotype of the 357th locus of the PGM2 gene is the TC peak diagram;

[0025] Figure 2 In the embodiment, the haplotype of the 357th locus of the PGM2 gene is the TT peak diagram. DETAILED DESCRIPTION

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The terms used in the specification of the application are for the purpose of describing specific embodiments only and are not intended to limit this application. The terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0027] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0028] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0029] Example: This example, through genome-wide association analysis of low dissolved oxygen tolerance in Litopenaeus vannamei, identified a locus associated with low dissolved oxygen tolerance. This locus, located within the PGM2 gene, harbors a T / C mutation, and individuals with the T / T genotype exhibit significantly higher low dissolved oxygen tolerance than individuals with the T / C genotype. Direct sequencing can be used to detect polymorphism at this SNP in a population of Litopenaeus vannamei, and the correlation between genotype frequency and low dissolved oxygen tolerance in the shrimp can be analyzed. In selective breeding for stress tolerance in Litopenaeus vannamei, this SNP can be used to prioritize T / T individuals as breeding parents, avoiding individuals with the T / C genotype, thereby improving selection efficiency and accelerating the breeding process.

[0030] The correlation analysis between the SNP site 357T>C in the PGM2 gene of Litopenaeus vannamei and hypoxia tolerance was carried out according to the following steps:

[0031] (A) Experimental sample preparation;

[0032] (B) Extraction of genomic DNA from Litopenaeus vannamei: In this example, a DNA nucleic acid extraction kit was used for rapid extraction;

[0033] (C) SNP screening of PGM2 gene in Litopenaeus vannamei, typing of site 357T>C;

[0034] (D) Correlation analysis between 357T>C genotype and low dissolved oxygen tolerance and assist in the breeding of low dissolved oxygen tolerant varieties;

[0035] The specific operations are as follows:

[0036] (A) Experimental sample preparation

[0037] 120 shrimp from different families, reared in a shared environment, were placed in a 3×3 m test pond. After temporary rearing, they were transferred to a 200 L water tank (actual water volume: 100 L) and subjected to a 72-hour hypoxic concentration (DL-50) stress experiment. The tank was covered with a membrane to isolate atmospheric oxygen. The shrimp consumed excess oxygen in the water through respiration. Micro-oxygenation was initiated when the dissolved oxygen (DO) concentration approached a pre-determined threshold (1.5 ± 0.1 mg / L) to maintain a stable hypoxic environment. DO concentrations were measured every two hours using a portable water quality meter. Half of the water was replaced every 12 hours, and fresh seawater was pre-filled with nitrogen to maintain hypoxia. The entire water exchange process was conducted in a closed environment. No feed was provided during the experiment. Mortality was observed every hour, and the survival time of each shrimp was recorded. Muscle tissue from the first 30 shrimp to die and the last 30 shrimp to die was stored in anhydrous ethanol for subsequent DNA extraction. The criterion for judging the death of shrimp is that if the shrimp lies on its side and does not respond to touch, it is considered dead. The experiment continues until all shrimp die.

[0038] (B) Extraction of genomic DNA from Litopenaeus vannamei

[0039] 30 mg of preserved shrimp muscle tissue was excised and placed in a sterile centrifuge tube. 200 μl of GA buffer and two sterile steel balls were added, and the sample was ground using a tissue grinder for 2 minutes. After grinding, 20 μl of Proteinase K (20 mg / ml) solution was added to the sample, and the sample was incubated at 56°C for 1 hour, with the sample shaken every 20 minutes for 15 seconds. 200 μl of Buffer GB was added, the sample was thoroughly inverted, and the sample was incubated at 70°C for 10 minutes. 20 μl of RNase A (10 mg / ml) was added, and the sample was allowed to stand at room temperature for 5 minutes. 200 μl of anhydrous ethanol was added, the sample was thoroughly inverted, and the solution was aspirated into an adsorption column. Centrifuged at 12,000 rpm for 30 seconds, the waste liquid was discarded, and the adsorption column was returned to the collection tube. 500 μl of Buffer GD was added to the adsorption column, and the sample was centrifuged at 12,000 rpm for 30 seconds. The waste liquid was discarded, and the adsorption column was returned to the collection tube. Add 600 μl of rinse buffer PW to the adsorption column and centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid in the collection tube and return the adsorption column to the collection tube. Repeat this step once. Return the adsorption column to the collection tube and centrifuge at 12,000 rpm for 2 minutes. Discard the waste liquid in the collection tube. Uncap the adsorption column and let it sit at room temperature for 5 minutes. Place the adsorption column in a sterile centrifuge tube and add 60 μl of elution buffer TE to the center of the adsorption membrane. Let it sit at room temperature for 5 minutes and then centrifuge at 12,000 rpm for 2 minutes. Aspirate the solution from the centrifuge tube and return it to the adsorption column. Let it sit at room temperature for 2 minutes and then centrifuge at 12,000 rpm for 2 minutes. Discard the adsorption column and retain the centrifuge tube and the solution within. This is the extracted DNA.

[0040] DNA integrity was analyzed by 1% agarose gel electrophoresis. The purity and concentration of the DNA solution were determined using a NanoDrop ND-2000 nucleic acid quantifier. DNA samples that passed the test were stored at -20°C for subsequent use. Acceptable DNA samples were characterized by a single, intact electrophoresis band with no tailing, a sample concentration greater than 50 ng / ul, and a sample purity (OD 260 / 280) between 1.6 and 2.0.

[0041] (C) SNP site screening and sequence site 357T>C typing of PGM2 gene in Litopenaeus vannamei;

[0042] The sequence of the PGM2 gene of Litopenaeus vannamei (Gen Bank Accession No. XM_070132845) was obtained from the NCBI website. Specific primer pairs, PGM2-F and PGM2-R, were designed using Premier 5.0 software. Genomic DNA of shrimp subjected to low dissolved oxygen stress was used as a template for PCR amplification using these primers. The PCR reaction system consisted of 50 μL of 25 μL Premix Taq™ (LATaq™ Version 2.0, TaKaRa), 17.5 μL of deionized water, 2.5 μL of forward primer, 2.5 μL of reverse primer, and 2.5 μL of genomic DNA template. The PCR amplification protocol was as follows: 94°C denaturation for 5 min; 33 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 1 min; after extension at 72°C for 10 min, the PCR product was refrigerated and transported to Guangzhou Shenggong Biotechnology Co., Ltd. for sequencing.

[0043] (D) Correlation analysis between T>C genotype and hypoxia tolerance

[0044] According to the sequencing results, the number of shrimps with each genotype T>C at site 357 in the PGM2 gene and the average survival time in the low dissolved oxygen stress experiment were counted. The chi-square test method of SPSS24.0 software was used to analyze the correlation between the number of T>C genotypes at site 357 of the PGM2 gene and the survival time. The statistical results are shown in Table 1.

[0045] The results showed that at site 357T>C, the distribution of the two different genotypes, TT and TC, was significantly associated with low dissolved oxygen tolerance (χ 2 =26.810, P=0.044). This suggests that the genotypic polymorphism at this locus significantly affects the tolerance of Litopenaeus vannamei to low dissolved oxygen, with individuals with the TT genotype exhibiting better tolerance to low dissolved oxygen than those with the TC genotype. During breeding and aquaculture, individuals with the TT genotype at locus 357T>C should be prioritized, while individuals with the TC genotype at locus 357T>C should be avoided.

[0046] Table 1 Results of association analysis of single nucleotide polymorphisms of gene PGM2

[0047]

[0048] Of the above number, 58 items were successfully sequenced.

[0049] Obviously, the embodiments described above are only some of the embodiments of the present application, rather than all of the embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions described in the aforementioned specific embodiments, or to make equivalent replacements for some of the technical features therein. Any equivalent structure made using the contents of the present application specification and the accompanying drawings, directly or indirectly used in other related technical fields, is also within the scope of patent protection of the present application.

Claims

1. A molecular marker associated with hypoxia stress tolerance in Litopenaeus vannamei, characterized by: A SNP molecular marker was cloned from the PGM2 gene. The SNP marker is a T or C at the 357th base from the 5' end of the first nucleotide sequence shown in the sequence table.

2. A primer pair for a molecular marker related to hypoxia stress resistance of Litopenaeus vannamei as claimed in claim 1, characterized in that: The primer pair is primer PGM2-F and primer PGM2-R, and the nucleotide sequences of the primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2: (1) SEQ ID NO.1: GCCACAGGTTTGCCAGAT; (2) SEQ ID NO. 2: CCAGAGGGTCAGTAAGTCG.

3. The primer pair for molecular markers related to the hypoxia stress resistance of Litopenaeus vannamei according to claim 2, characterized in that: Primers PGM2-F and PGM2-R were designed using Primer 5.0 software using the mRNA sequence of the PGM2 gene of Litopenaeus vannamei (Gen Bank Accession: NO. XM_070132845) as a template.

4. A primer pair for a molecular marker related to hypoxia stress resistance of Litopenaeus vannamei according to claim 2 or 3, characterized in that: The primer pair is used to perform PCR amplification on the segment where the SNP marker is located, and then the SNP marker is detected by sequencing, thereby determining the genotype of the SNP marker site of the tested Litopenaeus vannamei.

5. The molecular marker-assisted selection breeding method related to the low dissolved oxygen tolerance trait of Litopenaeus vannamei according to claim 2 or 3, characterized in that: (1) Sampling candidate populations of Penaeus vannamei breeding shrimp was performed. The sampling sites were selected from the tentacles or feet, which were less affected by the shrimp and could recover quickly. The sampled shrimp were individually marked using eye stalk rings or fluorescent markers. (2) directly sequencing the PCR products of the samples using the primers to obtain SNP site typing information; (3) Individuals with the TT genotype at the 357th SNP site in the PGM2 gene were selected as parents for breeding of low-oxygen-tolerant Litopenaeus vannamei for large-scale farming.

6. A method for screening molecular markers related to the low dissolved oxygen tolerance trait of Litopenaeus vannamei according to claim 2 or 3, characterized in that: Here are the steps: (1) Using the genomic DNA of the shrimp Litopenaeus vannamei after the low dissolved oxygen stress experiment as a template, PCR amplification was performed using the above-mentioned primer pair, namely primer PGM2-FP and primer PGM2-RP, to obtain an amplified fragment with a length of 300 bp; (2) sequencing the amplified fragment obtained in step (1) to obtain a partial sequence of the PGM2 gene, performing BLAST comparison on the sequence results to screen out base mutation sites, i.e., SNP sites; (3) The chi-square test of SPSS24.0 software was used to conduct a correlation analysis between the tolerance to low dissolved oxygen and the genotype of Penaeus vannamei.

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